1. Field of the Invention
The present invention relates to a positioning method for biochip spotting, and especially to a spotting positioning method that marks the positions of reference points on respective biochip substrates and makes feedback correction according to position information.
2. The Prior Arts
The word “biochip” originates in 1980s. Broadly speaking, it means to make bio-related molecules, such as gene, protein, carbohydrate and cell tissue, spotting on a substrate precisely for other uses with technology of micro area and high density. The manufacture of biochip uses several industry technologies and combines bio-medical technology with theories of molecular biology, analytic chemistry and bio-reaction to design for spotting the bio-molecules on the substrate materials of glass, silica plate and plastic (polymer material) to produce bio-chemistry analytic detection component. The advantages of biochip are that the biochip is capable of proceeding simultaneous, fast and mass bio-chemical detection on micro substrate area and their function or detective subjects are nuclear acid or protein, therefore it has a huge applicability on medical and bio-chemical analysis.
Biochips are divided into two categories according to their function. One is microarray chip (detective chip) and the other is micro-processing chip. The microarray chip, which is more common so far, is to spot bio-probe of high density on a micro area of a substrate to serve as a tool for mass assay and parallel analysis. Generally speaking, the size of a bio-probe on the biochip is not more than 200 μm in diameter, and the bio-probe is positioned in a particular groove or membrane of biochip with an automatic machine through setting parameters, including position in the matrix, length, density, and nucleotide sequence. Currently, there are three common spotting methods as:
(1) Contact spotting: The prepared bio-probes are directly spotted on a biochip with mechanical pins, which has the advantage of being simple and non-limited length of bio-probe. Two kinds of pins, solid pins and quill pins, can be used in this method.
(2) Non-contact jetting printing: The technology is similar to ink-jet printer, and it can print bio-probes on the surface of a biochip with a pattern of matrix.
(3) Photolithography: Photoetching is employed to synthesize bio-probes directly on a biochip, which is similar to the semiconductor technology.
Producing the “microarray chip” is to make some kinds of known specific bio-probes, such as protein and nucleotide, which are used as special detective elements spotted in rows on specific positions of the substrate in accordance with set order using the methods described above with a set of high precision mechanic equipment. A normal slide glass for microscope can be spotted with thousands or even dozens of thousands strictly aligned probes. Since the bio-probe put is very small, generally it can only be seen clearly under a microscope. In using the biochip, it is to make the sample being detected react with the bio-probe on the biochip to show the specific meaning of many actions among bio-molecules through the result of the reaction.
Since the bio-probes on the biochip are of a large number and a small volume, all the ways to maintain good respective relative position, distance and the specific position on the biochip will affect the result and interpretation of reaction afterward. Therefore, it becomes the important subject and direction of improving the yield of biochip production and success rate and reliability level of following experiments of biochip by controlling the spotting position of bio-probe on the biochip to form precise matrix on the specific area desired.
The spotting can proceed by batch according to the known technology. It can set the first spotting position of the first biochip substrate as the datum point, and then every spotting position of all the biochip substrates was determined according to the position of that datum point added with a constant distance to complete the spotting of the entire batch of biochip substrate sequentially. However, since the size of different chip of biochip substrate, which is usually glass or Nylon membrane, could have errors, and since the size of the bio-probe on the biochip is generally not more than 200 μm in diameter with the spacing distance in the order of micrometer, even a tiny error of substrate could result in the enlargement, reduction, distortion of spotting matrix or loss of matrix points, which in turn affects the following binding reaction, signal scanning, data analysis and increases the difficulty level and error rate of interpretation. It could even result in the entire batch of biochip being defective and unable to use. Furthermore, since the bio-probes are usually bio-material, which is not readily available, defective spotting will waste the precious bio-probes. Moreover, although distortion or error of biochip can be adjusted or treated with other methods, they all increase the time and cost of treatment massively.
An image alignment apparatus and method for microarray biochip produced by jet-printing technology is disclosed in Taiwan Patent No. 561070 which applies the principle of color jet printing system to the spotting of biochip. An image-taking device is arranged on a transportation device to allow the image-taking device to move relative to a printhead. An alignment sheet is employed to set the printhead to a previously determined position before the bio-reagents are jetted to a microarray biochip substrate in the form of micro liquid droplets.
However, the method is an improvement especially for spotting manner of non-contact jetting printing in which the printhead can hold still during the process of jetting but change the position of the substrate to proceed with spotting. It is totally different from the basic principle and method of spotting manner of contact spotting in which the spotting pin is at status of motion, and so it can not be used as well. Furthermore, it still has the same problem which happened often in the paper printing that the incorrect placement of paper will result in the shift and rotation of printing if the placement position of the biochip substrate during spotting is not proper. Moreover, the patent mentioned above needs to make the respective alignment of each other among printhead, alignment sheet and substrate to achieve more precise alignment, and so the procedure of alignment is more complicated and it is more difficult to control.